[Show abstract][Hide abstract] ABSTRACT: The oxygen-sensing prolyl hydroxylase domain proteins (PHDs) regulate cellular metabolism, but their role in neuronal metabolism during stroke is unknown. Here we report that PHD1 deficiency provides neuroprotection in a murine model of permanent brain ischemia. This was not due to an increased collateral vessel network. Instead, PHD1 -/- neurons were protected against oxygen-nutrient deprivation by reprogramming glucose metabolism. Indeed, PHD1 -/- neurons enhanced glucose flux through the oxidative pentose phosphate pathway by diverting glucose away from glycolysis. As a result, PHD1 -/- neurons increased their redox buffering capacity to scavenge oxygen radicals in ischemia. Intracerebroventricular injection of PHD1-antisense oligonucleotides reduced the cerebral infarct size and neurological deficits following stroke. These data identify PHD1 as a regulator of neuronal metabolism and a potential therapeutic target in ischemic stroke. How neuronal metabolism can be targeted to promote neuroprotection remains poorly understood. Quaegebeur et al. show that loss or inhibition of the oxygen sensor PHD1 protects against brain ischemia by shunting glucose into the oxidative pentose phosphate pathway, thereby improving oxygen radical detoxification and securing redox homeostasis.
[Show abstract][Hide abstract] ABSTRACT: Endothelial cells (ECs) are plastic cells that can switch between growth states with different bioenergetic and biosynthetic requirements. Although quiescent in most healthy tissues, ECs divide and migrate rapidly upon proangiogenic stimulation. Adjusting endothelial metabolism to the growth state is central to normal vessel growth and function, yet it is poorly understood at the molecular level. Here we report that the forkhead box O (FOXO) transcription factor FOXO1 is an essential regulator of vascular growth that couples metabolic and proliferative activities in ECs. Endothelial-restricted deletion of FOXO1 in mice induces a profound increase in EC proliferation that interferes with coordinated sprouting, thereby causing hyperplasia and vessel enlargement. Conversely, forced expression of FOXO1 restricts vascular expansion and leads to vessel thinning and hypobranching. We find that FOXO1 acts as a gatekeeper of endothelial quiescence, which decelerates metabolic activity by reducing glycolysis and mitochondrial respiration. Mechanistically, FOXO1 suppresses signalling by MYC (also known as c-MYC), a powerful driver of anabolic metabolism and growth. MYC ablation impairs glycolysis, mitochondrial function and proliferation of ECs while its EC-specific overexpression fuels these processes. Moreover, restoration of MYC signalling in FOXO1-overexpressing endothelium normalizes metabolic activity and branching behaviour. Our findings identify FOXO1 as a critical rheostat of vascular expansion and define the FOXO1-MYC transcriptional network as a novel metabolic checkpoint during endothelial growth and proliferation.
[Show abstract][Hide abstract] ABSTRACT: Chloroquine is used clinically as an autophagy blocker to potentiate anticancer treatments. However, whether chloroquine acts solely through autophagy-dependent and cancer cell autonomous mechanisms has remained elusive. In a recent study we found that chloroquine reduced intratumoral hypoxia and metastasis, while improving chemotherapy response, largely through an autophagy-independent, NOTCH1-reliant mechanism of tumor vessel normalization.
[Show abstract][Hide abstract] ABSTRACT: Background & Aims
Histamine sensitizes the nociceptor TRPV1 and has been shown to contribute to visceral hypersensitivity in animals. We investigated the role of TRPV1 in irritable bowel syndrome (IBS) and evaluated if an antagonist of histamine receptor H1 (HRH1) could reduce symptoms of patients in a randomized placebo controlled trial.
Using live calcium imaging, we compared activation of submucosal neurons by the TRPV1 agonist capsaicin in rectal biopsies collected from 9 patients with IBS (ROME 3 critera) and 15 healthy subjects. Sensitization of TRPV1 by histamine, its metabolite imidazole acetaldehyde, and supernatants from biopsies was assessed by calcium imaging of mouse dorsal root ganglion neurons. We then performed a double-blind trial of patients with IBS (mean age, 31 years; range, 18–65 years; 34 female). After a 2 week run-in period, subjects were randomly assigned to groups given either the HRH1 antagonist ebastine (20 mg/day; n=28) or placebo (n=27) for 12 weeks. Rectal biopsies were collected, barostat studies were performed, and symptoms were assessed (using the validated gastrointestinal symptom rating scale) before and after the 12 week period. Patients were followed for an additional 2 weeks. Abdominal pain, symptom relief, and health-related quality of life were assessed on a weekly basis. The primary endpoint of the study was the effect of ebastine on the symptom score evoked by rectal distension.
TRPV1 responses of submucosal neurons from patients with IBS were potentiated compared to those of healthy volunteers. Moreover, TRPV1 responses of submucosal neurons from healthy volunteers could be potentiated by their pre-incubation with histamine; this effect was blocked by the HRH1 antagonist pyrilamine. Supernatants from rectal biopsies from patients with IBS, but not from the healthy volunteers, sensitized TRPV1 in mouse nociceptive dorsal root ganglion neurons via HRH1; this effect could be reproduced by histamine and imidazole acetaldehyde. Compared to subjects given placebo, those given ebastine had reduced visceral hypersensitivity, increased symptom relief (ebastine 46% vs placebo 13%; P=.024) and reduced abdominal pain scores (ebastine 39±23 vs placebo 62±22, P=.0004).
In studies of rectal biopsies from patients, we found that HRH1-mediated sensitization of TRPV1 is involved in IBS. Ebastine, an antagonist of HRH1, reduced visceral hypersensitivity, symptoms, and abdominal pain in patients with IBS. Inihbitors of this pathway might be developed as a new treatment approach for IBS. ClinicalTrials.gov no: NCT01144832
[Show abstract][Hide abstract] ABSTRACT: Study hypothesis:
Placental growth factor (PGF) is expressed in the developing mouse brain and contributes to vascularization and vessel patterning.
PGF is dynamically expressed in fetal mouse brain, particularly forebrain, and is essential for normal cerebrovascular development.
What is known already:
PGF rises in maternal plasma over normal human and mouse pregnancy but is low in many women with the acute onset hypertensive syndrome, preeclampsia (PE). Little is known about the expression of PGF in the fetus during PE. Pgf(-/-) mice appear normal but recently cerebral vascular defects were documented in adult Pgf(-/-) mice.
Study design, samples/materials, methods:
Here, temporal-spatial expression of PGF is mapped in normal fetal mouse brains and cerebral vasculature development is compared between normal and congenic Pgf(-/-) fetuses to assess the actions of PGF during cerebrovascular development. Pgf/PGF, Vegfa/VEGF, Vegf receptor (Vegfr)1 and Vegfr2 expression were examined in the brains of embryonic day (E)12.5, 14.5, 16.5 and 18.5 C57BL/6 (B6) mice using quantitative PCR and immunohistochemistry. The cerebral vasculature was compared between Pgf(-/-) and B6 embryonic and adult brains using whole mount techniques. Vulnerability to cerebral ischemia was investigated using a left common carotid ligation assay.
Main results and the role of chance:
Pgf/PGF and Vegfr1 are highly expressed in E12.5-14.5 forebrain relative to VEGF and Vegfr2. Vegfa/VEGF is relatively more abundant in hindbrain. PGF and VEGF expression were similar in midbrain. Delayed hindbrain vascularization was seen at E10.5 and 11.5 in Pgf(-/-) brains. At E14.5, Pgf(-/-) circle of Willis showed unilateral hypoplasia and fewer collateral vessels, defects that persisted postnatally. Functionally, adult Pgf(-/-) mice experienced cerebral ischemia after left common carotid arterial occlusion while B6 mice did not.
Limitations, reasons for caution:
Since Pgf(-/-) mice were used, consequences of complete absence of maternal and fetal PGF were defined. Therefore, the effects of maternal versus fetal PGF deficiency on cerebrovascular development cannot be separated. However, as PGF was strongly expressed in the developing brain at all timepoints, we suggest that local PGF has a more important role than distant maternal or placental sources. Full PGF loss is not expected in PE pregnancies, predicting that the effects of PGF deficiency identified in this model will be more severe than any effects in PE-offspring.
Wider implications of the findings:
These studies provoke the question of whether PGF expression is decreased and cerebral vascular maldevelopment occurs in fetuses who experience a preeclamptic gestation. These individuals have already been reported to have elevated risk for stroke and cognitive impairments.Large scale data.
N/a study funding and competing interests:
This work was supported by awards from the Natural Sciences and Engineering Research Council, the Canada Research Chairs Program and the Canadian Foundation for Innovation to BAC and by training awards from the Universidade Federal de Pernambuco and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil to RLL; Queen's University to VRK and the Canadian Institutes of Health Research to MTR. The work of PC is supported by the Belgian Science Policy BELSPO - IUAP7/03, Structural funding by the Flemish Government - Methusalem funding, and the Flemish Science Fund - FWO grants. There were no competing interests.
Full-text · Article · Dec 2015 · Molecular Human Reproduction
[Show abstract][Hide abstract] ABSTRACT: In endothelial cells, binding of vascular endothelial growth factor (VEGF) to the receptor VEGFR2 activates multiple signaling pathways that trigger processes such as proliferation, survival, and migration that are necessary for angiogenesis. VEGF-bound VEGFR2 becomes internalized, which is a key step in the proangiogenic signal. We showed that the urokinase plasminogen activator receptor (uPAR) interacted with VEGFR2 and described the mechanism by which this interaction mediated VEGF signaling and promoted angiogenesis. Knockdown of uPAR in human umbilical vein endothelial cells (HUVECs) impaired VEGFR2 signaling, and uPAR deficiency in mice prevented VEGF-induced angiogenesis. Upon exposure of HUVECs to VEGF, uPAR recruited the low-density lipoprotein receptor-related protein 1 (LRP-1) to VEGFR2, which induced VEGFR2 internalization. Thus, the uPAR-VEGFR2 interaction is crucial for VEGF signaling in endothelial cells.
No preview · Article · Nov 2015 · Science Signaling
[Show abstract][Hide abstract] ABSTRACT: Tumour angiogenesis has long been recognised as a target for anti-cancer therapy. The current approach of inhibiting the VEGF pathway has shown benefit in the clinic, though less than anticipated. We recently documented that glycolytic metabolism in endothelial cells (ECs) fuels angiogenesis, rendering it a possible target for inhibiting vascular growth in pathological conditions. More recently, we reported that the oxidation of fatty acids (FA) is irreplaceable for EC proliferation by providing carbons for de novo nucleotide synthesis. Furthermore, ECs are rather unique in this respect, creating novel therapeutic opportunities. Here, we review and compare the current understanding of FA utilisation in ECs and tumour cells (TCs).
No preview · Article · Nov 2015 · Critical reviews in oncology/hematology
[Show abstract][Hide abstract] ABSTRACT: Hematopoietic stem cells (HSCs) in the fetal liver (FL) unlike adult bone marrow (BM) proliferate extensively, posing different metabolic demands. However, metabolic pathways responsible for the production of energy and cellular building blocks in FL HSCs have not been described. Here, we report that FL HSCs use oxygen dependent energy generating pathways significantly more than their BM counterparts. RNA-Seq analysis of E14.5 FL versus BM derived HSCs identified increased expression levels of genes involved in oxidative phosphorylation (OxPhos) and the citric acid cycle (TCA). We demonstrated that FL HSCs contain more mitochondria than BM HSCs, which resulted in increased levels of oxygen consumption and reactive oxygen species (ROS) production. Higher levels of DNA repair and antioxidant pathway gene expression may prevent ROS-mediated (geno)toxicity in FL HSCs. Thus, we here for the first time highlight the underestimated importance of oxygen dependent pathways for generating energy and building blocks in FL HSCs.
Full-text · Article · Nov 2015 · Stem Cell Research
[Show abstract][Hide abstract] ABSTRACT: Introduction: Prolyl hydroxylase domain (PHD) deletion results in the stabilization
of hypoxia inducible factor (HIF), a key compound in the cellular response
following hypoxia which has been linked to cancer development. We previously
showed that PHD inhibition results in an increased expression of liver progenitor
cells (LPCs) which coincided with a phenotypic switch from hepatocellular car-
cinoma (HCC) to hepato-cholangiocarcinoma in diethylnitrosamine (DEN)induced
Aims & Methods: In this study, we investigated if the effect of PHD2 inhibition
on the expression of markers for hypoxia and LPCs occurs during the initiation
and development of HCC following DEN treatment. PHD2 haplodeficient
(PHD2+/-) and wild type (WT) mice were weekly injected with 35mg/kg DEN
and euthanized after 5, 10, 15 and 17.5 weeks of DEN induction, before tumor
nodules could form1. Livers were prelevated and analyzed for the expression of
downstream targets of HIF (Vascular endothelial growth factor alpha, Vegfa and
Glucose transporter 1, Glut1) and LPC markers (cytokeratin 7, CK7; cytokeratin
19, CK19; prominin1, Prom1 and epithelial cell adhesion molecule, Epcam) using
qPCR and immunohistochemistry. The results were linked to neoplastic transformation
of hepatocytes determined by reticulin staining. Results were significant
at p <0.05.
Results: Vegfa and Glut1 RNA expression were significantly upregulated after
17.5 weeks of DEN induction in WT and PHD
higher increase for Glut1 in PHD2+/- mice with a significantly
mice. Prom1 and CK19 expression followed these results with a significant increase after 17.5 weeks of DEN induction, however without differences between PHD2+/- and WT mice. For CK7 and
Epcam, the RNA expression did not change during early hepatocarcinogenesis.
Immunohistochemistry revealed an increased CK19 immunoreactivity after 15
weeks of DEN induction compared to earlier time points in PHD
was mostly caused by increased cytoplasmic expression in hepatocytes rather
than ductular expansion. The intracellular domain of Epcam was significantly
more expressed after 17.5 weeks of DEN in PHD2+/- mice compared to WT mice.
Neoplastic transformation was limited to some reticulin free cells after 15 and
17.5 weeks of DEN in both PHD2+/-and WT mice.
Conclusion: These results show an increased expression of LPC characteristics,
coinciding with augmented expression of hypoxic markers during early HCCdevelopment. However, the effects of PHD2 haplodeficiency were limited to
non-existing. Based on this and our previous reports1,2
we conclude that induction of the hypoxic response affects the expression of LPC characteristics and
tumor phenotype not in an early event but coincides with the occurrence of neoplasticity.
1. Heindryckx, F et al. Effect of prolyl hydroxylase domain-2 haplodeficiency
on the hepatocarcinogenesis in mice. JHep 2012.
2. Bogaerts, E et al. Time dependent effect of hypoxia on tumor progression
and liver progenitor cell markers in primary liver tumors. Plos One 2015.
[Show abstract][Hide abstract] ABSTRACT: Cell division is a metabolically demanding process, requiring the production of large amounts of energy and biomass. Not surprisingly therefore, a cell's decision to initiate division is co-determined by its metabolic status and the availability of nutrients. Emerging evidence reveals that metabolism is not only undergoing substantial changes during the cell cycle, but it is becoming equally clear that metabolism regulates cell cycle progression. Here, we overview the emerging role of those metabolic pathways that have been best characterized to change during or influence cell cycle progression. We then studied how Notch signaling, a key angiogenic pathway that inhibits endothelial cell (EC) proliferation, controls EC metabolism (glycolysis) during the cell cycle.
[Show abstract][Hide abstract] ABSTRACT: Cardiomyopathy is among the leading causes of death from systemic sclerosis (SSc). Urokinase-type plasminogen activator receptor (uPAR)-deficient mice have been recently reported to display important histopathological hallmarks of SSc, including dermal fibrosis, reduced dermal capillary density, and pulmonary fibrosis. Here, we investigated whether uPAR-deficient mice could display the histopathological features of SSc-related cardiomyopathy.
Ventricular myocardial specimens from uPAR-deficient and wild-type mice at 12 and 24 weeks of age were analysed by both light microscopy and transmission electron microscopy. Picrosirius red staining and hydroxyproline content of myocardial specimens were quantified. Myofibroblast and microvessel counts were determined by immunofluorescence for α-smooth muscle actin and CD31, respectively. Endothelial cell apoptosis was assessed by a combined TUNEL/CD31 immunofluorescence assay. Expression of uPAR in human SSc and control ventricular myocardial autopsy specimens was determined by immunohistochemistry.
The myocardium of 24-week-old uPAR-deficient mice displayed focal ischaemic lesions with cardiomyocyte hypertrophy, myofibril rarefaction and contraction band necrosis. At 24 weeks of age, interstitial and perivascular collagen deposition and myofibroblast counts were significantly greater in myocardial tissue of uPAR-deficient mice than in wild-type mice. In uPAR-deficient mice, myocardial fibrosis was paralleled by microvascular endothelial cell apoptosis and reduced capillary density. uPAR expression was significantly downregulated in the myocardium of patients with SSc.
Typical histopathological features of SSc-related cardiomyopathy are mimicked by uPAR-deficient mice. The downregulation of uPAR in the myocardium of patients with SSc may suggest similar underlying pathogenetic mechanisms. uPAR-deficient mice could be used as a preclinical model to study the mechanisms and therapeutic approaches of myocardial involvement in SSc.
Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.
No preview · Article · Aug 2015 · Annals of the Rheumatic Diseases
[Show abstract][Hide abstract] ABSTRACT: During vessel sprouting, endothelial "tip" cells migrate at the forefront, while the endothelial "stalk" cells elongate the sprout; endothelial "phalanx" cells line quiescent vessels. Tip and stalk cells can dynamically switch phenotypes under the control of VEGF and Notch signaling. Novel findings now show that in addition to signaling cascades, metabolism co-regulates the formation of the new vasculature. Recent studies demonstrated that endothelial cells (ECs) rely primarily on glycolysis for ATP production, that glycolysis is further enhanced in angiogenic ECs, and that the key glycolytic regulator PFKFB3 co-determines angiogenesis by controlling the balance of tip versus stalk cells and promoting a migratory tip cell phenotype. On the other hand, fatty acid oxidation regulates endothelial stalk cell proliferation by providing carbon sources for biosynthetic processes, more particularly for de novo nucleotide synthesis for DNA replication. Here, we overview the current understanding of the various metabolic pathways in ECs and their impact on vessel formation in health and disease. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
No preview · Article · Aug 2015 · Microcirculation (New York, N.Y.: 1994)
[Show abstract][Hide abstract] ABSTRACT: Angiogenesis has been traditionally studied by focusing on growth factors and other proangiogenic signals, but endothelial cell (EC) metabolism has not received much attention. Nonetheless, glycolysis, one of the major metabolic pathways that converts glucose to pyruvate, is required for the phenotypic switch from quiescent to angiogenic ECs. During vessel sprouting, the glycolytic activator PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase-3) promotes vessel branching by rendering ECs more competitive to reach the tip of the vessel sprout, whereas fatty acid oxidation selectively regulates proliferation of endothelial stalk cells. These studies show that metabolic pathways in ECs regulate vessel sprouting, more importantly than anticipated. This review discusses the recently discovered role of glycolysis and fatty acid oxidation in vessel sprouting. We also highlight how metabolites can influence EC behavior as signaling molecules by modulating posttranslational modification.
No preview · Article · Jul 2015 · The Cancer Journal
[Show abstract][Hide abstract] ABSTRACT: The metabolism of endothelial cells during vessel sprouting remains poorly studied. Here we report that endothelial loss of CPT1A, a rate-limiting enzyme of fatty acid oxidation (FAO), causes vascular sprouting defects due to impaired proliferation, not migration, of human and murine endothelial cells. Reduction of FAO in endothelial cells did not cause energy depletion or disturb redox homeostasis, but impaired de novo nucleotide synthesis for DNA replication. Isotope labelling studies in control endothelial cells showed that fatty acid carbons substantially replenished the Krebs cycle, and were incorporated into aspartate (a nucleotide precursor), uridine monophosphate (a precursor of pyrimidine nucleoside triphosphates) and DNA. CPT1A silencing reduced these processes and depleted endothelial cell stores of aspartate and deoxyribonucleoside triphosphates. Acetate (metabolized to acetyl-CoA, thereby substituting for the depleted FAO-derived acetyl-CoA) or a nucleoside mix rescued the phenotype of CPT1A-silenced endothelial cells. Finally, CPT1 blockade inhibited pathological ocular angiogenesis in mice, suggesting a novel strategy for blocking angiogenesis.